Response Of Mammary Epithelial Cells Research Articles

MyD88 deficiency in mammary epithelial cells attenuates lipopolysaccharide (LPS)-induced mastitis in mice

Lactation mastitis is a debilitating inflammatory mammary disease in postpartum animals. Myeloid differentiation primary response protein MyD88 is the key downstream adapter for innate pattern recognition receptor toll-like receptor 4 (TLR4), which plays an important role in inflammation. However, the specific role of MyD88 in mammary epithelial cells in the progression of mastitis has not been investigated. In this study, lipopolysaccharide (LPS)-induced mouse mastitis model was used and cytokines such as Tnf-α, Il-1β, Il-6, Cxcl1, Cxcl2 and Ccl2 were significantly increased in inflammatory mammary gland as shown by real time-qPCR. However, the mice with MyD88-deficienet in mammary epithelial cells (cKO) showed a reduction in the expression of Tnf-α, Il-1β, Il-6, Cxcl1 and Cxcl2 in mammary gland compared with control mice, when subjected to LPS induced mastitis. Immunohistochemical staining of cleaved caspase-3 showed that the cell apoptosis induced by inflammation were decreased in MyD88 cKO mice. Furthermore, there were significantly fewer infiltrating inflammatory cells in alveolar lumen of MyD88 cKO mice, including Ly6G-positive neutrophils and F4/80-positive macrophages. RNA-seq in LPS treated mammary glands showed that MyD88 cKO mice had significantly downregulated inflammation-related genes and upregulated genes related to anti-inflammation processes and lipid metabolism compared with control mice. Thus, these results demonstrate that MyD88 in mammary epithelial cells is essential for mastitis progression. And this study not only has important implications for understanding the innate immune response in mammary epithelial cells, but also potentially helps the development of new therapeutic drugs for treating mastitis.

Breast Cancer Cells Exhibit Mesenchymal-Epithelial Plasticity Following Dynamic Modulation of Matrix Stiffness.

Mesenchymal-epithelial transition (MET) is essential for tissue and organ development and is thought to contribute to cancer by enabling the establishment of metastatic lesions. Despite its importance in both health and disease, there is a lack of in vitro platforms to study MET and little is known about the regulation of MET by mechanical cues. Here, hyaluronic acid-based hydrogels with dynamic and tunable stiffnesses mimicking that of normal and tumorigenic mammary tissue are synthesized. The platform is then utilized to examine the response of mammary epithelial cells and breast cancer cells to dynamic modulation of matrix stiffness. Gradual softening of the hydrogels reduces proliferation and increases apoptosis of breast cancer cells. Moreover, breast cancer cells exhibit temporal changes in cell morphology, cytoskeletal organization, and gene expression that are consistent with mesenchymal-epithelial plasticity as the stiffness of the matrix is reduced. A reduction in matrix stiffness attenuates the expression of integrin-linked kinase, and inhibition of integrin-linked kinase impacts proliferation, apoptosis, and gene expression in cells cultured on stiff and dynamic hydrogels. Overall, these findings reveal intermediate epithelial/mesenchymal states as cells move along a matrix stiffness-mediated MET trajectory and suggest an important role for matrix mechanics in regulating mesenchymal-epithelial plasticity.

Soluble CD14 produced by bovine mammary epithelial cells modulates their response to full length LPS

Bovine mastitis remains a major disease in cattle world-wide. In the mammary gland, mammary epithelial cells (MEC) are sentinels equipped with receptors allowing them to detect and respond to the invasion by bacterial pathogens, in particular Escherichia coli. Lipopolysaccharide (LPS) is the major E. coli motif recognized by MEC through its interaction with the TLR4 receptor and the CD14 co-receptor. Previous studies have highlighted the role of soluble CD14 (sCD14) in the efficient recognition of LPS molecules possessing a full-length O-antigen (LPSS). We demonstrate here that MEC are able to secrete CD14 and are likely to contribute to the presence of sCD14 in milk. We then investigated how sCD14 modulates and is required for the response of MEC to LPSS. This study highlights the key role of sCD14 for the full activation of the Myd88-independent pathway by LPSS. We also identified several lncRNA that are activated in MEC in response to LPS, including one lncRNA showing homologies with the mir-99a-let-7c gene (MIR99AHG). Altogether, our results show that a full response to LPS by mammary epithelial cells requires sCD14 and provide detailed information on how milk sCD14 can contribute to an efficient recognition of LPS from coliform pathogens.

Regulation of Inflammatory Responses of Cow Mammary Epithelial Cells through MAPK Signaling Pathways of IL-17A Cytokines.

The aim of this study is to explore the mechanism of IL-17A infection in the development of bacterial mastitis in dairy cows. In this study, RT-qPCR and ELISA were used to measure the promoting effect of IL-17A on the generation of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and chemokine (IL-8). In addition, Western blot (WB) was applied to measure the influences of IL-17A on the inflammation-related ERK and p38 proteins in the MAPK pathways. The results show that under the stimulation of LPS on cow mammary epithelial cells (CMECs), cytokines IL-1β, IL-6, IL-8, TNF-α, and IL-17A will exhibit significantly increased expression levels (p < 0.05). With inhibited endogenous expression of IL-17A, cytokines IL-1β, IL-6, IL-8, and TNF-α will present reduced genetic expression (p < 0.01), with reduced phosphorylation levels of ERK and p38 proteins in the MAPK signaling pathways (p < 0.001). Upon the exogenous addition of the IL-17A cytokine, the genetic expression of cytokines IL-1β, IL-6, IL-8, and TNF-α will increase (p < 0.05), with increased phosphorylation levels of the ERK and p38 proteins in the MAPK signaling pathways (p < 0.001). These results indicate that under the stimulation of CMECs with LPS, IL-17A can be expressed together with relevant inflammatory cytokines. Meanwhile, the inflammatory responses of mammary epithelial cells are directly proportional to the expression levels of IL-17A inhibited alone or exogenously added. In summary, this study shows that IL-17A could be used as an important indicator for assessing the bacterial infections of mammary glands, indicating that IL-17A could be regarded as one potential therapeutic target for mastitis.

PAPAS promotes differentiation of mammary epithelial cells and suppresses breast carcinogenesis

Extensive remodeling of the female mammary epithelium during development and pregnancy has been linked to cancer susceptibility. The faithful response of mammary epithelial cells (MECs) to hormone signaling is key to avoiding breast cancer development. Here, we show that lactogenic differentiation of murine MECs requires silencing of genes encoding ribosomal RNA (rRNA) by the antisense transcript PAPAS. Accordingly, knockdown of PAPAS derepresses rRNA genes, attenuates the response to lactogenic hormones, and induces malignant transformation. Restoring PAPAS levels in breast cancer cells reduces tumorigenicity and lung invasion and activates many interferon-regulated genes previously linked to metastasis suppression. Mechanistically, PAPAS transcription depends on R-loop formation at the 3' end of rRNA genes, which is repressed by RNase H1 and replication protein A (RPA) overexpression in breast cancer cells. Depletion of PAPAS and upregulation of RNase H1 and RPA in human breast cancer underpin the clinical relevance of our findings.

Free Fatty Acids Induce Apoptosis of Mammary Epithelial Cells of Ketotic Dairy Cows via the Mito-ROS/NLRP3 Signaling Pathway.

Early lactation increases metabolic stress in ketotic dairy cows, leading to mitochondrial damage, apoptosis, and inflammatory response in mammary epithelial cells. The pyrin domain 3 (NLRP3) pathway involving the mitochondrial reactive oxygen species (Mito-ROS)-induced nucleotide-binding oligomerization domain-like receptor has been recognized as a key mechanism in this inflammatory response and cell apoptosis. This study aimed to elucidate the underlying regulatory mechanism of Mito-ROS-NLRP3 pathway-mediated mammary epithelial cell apoptosis in dairy cows with ketosis. Mitochondrial damage and cellular apoptotic program and NLRP3 inflammasome activation were observed in the mammary gland of ketotic cows. Similar damage was detected in MAC-T cells treated with exogenous fatty acids (FFAs). However, NLRP3 inhibitor MCC950 pretreatment or Mito-ROS scavenging by MitoTEMPO attenuated apoptosis in FFA-induced MAC-T cells by inhibiting the NLRP3 inflammasome pathway. These findings reveal that the Mito-ROS-NLRP3 pathway activation is a potent mechanism underlying mammary epithelial cell apoptosis in response to metabolic stress in ketotic dairy cows, which further contributes to reduced milk yield.

Genetics, environmental stress, and amino acid supplementation affect lactational performance via mTOR signaling pathway in bovine mammary epithelial cells.

Mammary glands are known for their ability to convert nutrients present in the blood into milk contents. In cows, milk synthesis and the proliferation of cow mammary epithelial cells (CMECs) are regulated by various factors, including nutrients such as amino acids and glucose, hormones, and environmental stress. Amino acids, in particular, play a crucial role in regulating cell proliferation and casein synthesis in mammalian epithelial cells, apart from being building blocks for protein synthesis. Studies have shown that environmental factors, particularly heat stress, can negatively impact milk production performance in dairy cattle. The mammalian target of rapamycin complex 1 (mTORC1) pathway is considered the primary signaling pathway involved in regulating cell proliferation and milk protein and fat synthesis in cow mammary epithelial cells in response to amino acids and heat stress. Given the significant role played by the mTORC signaling pathway in milk synthesis and cell proliferation, this article briefly discusses the main regulatory genes, the impact of amino acids and heat stress on milk production performance, and the regulation of mTORC signaling pathway in cow mammary epithelial cells.

Mitochondrial Calcium Uniporter Regulator 1 (MCUR1) Relieves Mitochondrial Damage Induced by Lipopolysaccharide by Mediating Mitochondrial Ca2+ Homeostasis in Bovine Mammary Epithelial Cells

The metabolic stress triggered by negative energy balance after calving induces mitochondrial damage of bovine mammary epithelial cells. Mitochondrial calcium uniporter regulator 1 (MCUR1) is a key protein-coding gene that mediates mitochondrial calcium ion (Ca2+) uptake and plays an important role in mediating homeostasis of mitochondria. The aim of the present study was to elucidate the effects of MCUR1-mediated Ca2+ homeostasis on mitochondria of bovine mammary epithelial cells in response to an inflammatory challenge with lipopolysaccharide (LPS). Exogenous LPS resulted in upregulation of the MCUR1 mRNA and protein abundance, mitochondrial Ca2+ content, and mitochondrial reactive oxygen species (Mito-ROS) content while decreasing mitochondrial membrane potential, causing mitochondrial damage, and increasing the rate of apoptosis. Ryanodine pretreatment attenuated the upregulation of the mitochondrial Ca2+ content and Mito-ROS content induced by LPS. Overexpression of MCUR1 increased the mitochondrial Ca2+ content and Mito-ROS content, while it decreased mitochondrial membrane potential, damaged mitochondria, and induced cell apoptosis. In addition, knockdown of MCUR1 by small interfering RNA attenuated LPS-induced mitochondrial dysfunction by inhibiting mitochondrial Ca2+ uptake. Our results revealed that exogenous LPS induces MCUR1-mediated mitochondrial Ca2+ overload in bovine mammary epithelial cells, which leads to mitochondrial injury. Thus, MCUR1-mediated Ca2+ homeostasis may be a potential therapeutic target against mitochondrial damage induced by metabolic challenges in bovine mammary epithelial cells.

Distinct breast milk microbiota, cytokine, and adipokine profiles are associated with infant growth at 12 months: an in vitro host-microbe interaction mechanistic approach.

Breast milk (BM) is important for adequate infant development, and it contains bioactive compounds, such as bacteria, cytokines and some adipokines which play a role in infant microbial, metabolic, and immunological maturation. However, little is known about its impact on growth and development in early life. The objective of this study was to evaluate the impact of milk microbiota, cytokine, and adipokine profiles on the risk of overweight at 12 months of life to find the possible mechanisms of host-microbe interactions. In this study, BM samples from 100 healthy women collected during 15 d after birth were included. BM microbiota was analysed by 16S rRNA gene sequencing, and cytokine and adipokine levels were measured by the Luminex approach. In addition, infant weight and length were recorded during the first 12 months and z-scores were obtained according to the WHO databases. Infants were classified as risk of overweight (ROW) and no-risk of overweight (NOROW) based on their body mass index z-score (BMIZ) and infant weight-for-length z-score (WLZ) at 12 months. In order to study host-microbe interactions, epithelial intestinal and mammary cell lines were exposed to milk microbes to assess the host response by interleukin (IL)-6 production as a potential inflammatory marker. BM was dominated by Staphylococcus and Streptococcus genera, and the most abundant cytokines were IL-6 and IL-18. Leptin levels were positively correlated with the pregestational body mass index (BMI). Higher relative abundance of the Streptococcus genus was associated with higher IL-10 and higher relative abundance of the Bifidobacterium genus was associated with lower IL-6 concentrations in milk. Infant WLZ at 12 months could be partially predicted by Streptococcus genus proportions and IL-10 and IL-18 levels in BM. BM microbiota significantly induced cytokine responses in mammary epithelial cells. Higher levels of IL-6 production were observed in mammary cells exposed to BM microbiota from mothers with ROW offspring compared to mothers with NOROW offspring. In conclusion, BM microbiota is related to the cytokine profile. IL-10 and IL-18 levels and the abundance of the Streptococcus genus could affect early infant development. Further research is needed to clarify the specific impact of BM microbiota and cytokines on infant growth and the risk of overweight.

Correction: Jiao et al. Bta-miR-223 Targeting the RHOB Gene in Dairy Cows Attenuates LPS-Induced Inflammatory Responses in Mammary Epithelial Cells. Cells 2022, 11, 3144

In the original publication [...].

Novel cryo-tomography workflow reveals nanometer-scale responses of epithelial cells to matrix stiffness and dimensionality.

Matrix stiffness and dimensionality have been shown to be major determinants of cell behavior. However, a workflow for examining nanometer-scale responses of the associated molecular machinery is not available. Here, we describe a comprehensive, quantitative workflow that permits the analysis of cells responding to mechanical and dimensionality cues in their native state at nanometer scale by cryogenic electron tomography. Using this approach, we quantified distinct cytoskeletal nanoarchitectures and vesicle phenotypes induced in human mammary epithelial cells in response to stiffness and dimensionality of reconstituted basement membrane. Our workflow closely recapitulates the microenvironment associated with acinar morphogenesis and identified distinct differences in situ at nanometer scale. Using drug treatment, we showed that molecular events and nanometer-scale rearrangements triggered by engagement of apical cell receptors with reconstituted basement membrane correspond to changes induced by reduction of cortical tension. Our approach is fully adaptable to any kind of stiffness regime, extracellular matrix composition, and drug treatment.

Bta-miR-223 Targeting the RHOB Gene in Dairy Cows Attenuates LPS-Induced Inflammatory Responses in Mammary Epithelial Cells.

Bovine mammary epithelial cells (bMECs) are part of the first line of defense against pathogens. In recent studies, bta-miR-223 has been reported to activate congenital and innate immunity against inflammatory damage during the pathogenesis of mastitis in dairy cows. The purpose of this study was to identify the regulatory mechanism of bta-miR-223 and its downstream target genes in inflammatory bMECs. A double luciferase reporter gene assay demonstrated that ras homolog family member B (RHOB) was the target gene of bta-miR-223. To further elucidate the role of bta-miR-223 in congenital immune responses, bta-miR-223 mimics (mimic/inhibitor) were transfected into bMECs stimulated with lipopolysaccharide (LPS), which activates the Toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) signaling pathway. Real-time quantitative PCR (qPCR) and Western blot were used to detect the expression of related genes and proteins, and enzyme-linked immunosorbent assay (ELISA) was used to detect secreted inflammatory factors. Results showed that bta-miR-223 expression during inflammation in bMECs reduced the secretion of inflammatory factors by targeting RHOB and deactivation of NF-κB gene activity. Silencing RHOB inhibited LPS-induced inflammatory response in bMECs. Overall, bta-miR-223 attenuated LPS-induced inflammatory response, and acted as a negative feedback regulator via targeting RHOB, providing a novel avenue for mastitis treatment.

Inhibiting nuclear factor erythroid 2 related factor 2-mediated autophagy in bovine mammary epithelial cells induces oxidative stress in response to exogenous fatty acids

BackgroundIn early lactation, bovine mammary epithelial cells undergo serious metabolic challenges and oxidative stress both of which could be alleviated by activation of autophagy. Nuclear factor erythroid 2 related factor 2 (NFE2L2), a master regulator of cellular redox homeostasis, plays an important role in the regulation of autophagy and oxidative stress. Thus, the objective of this study was to investigate the role of NFE2L2-mediated autophagy on oxidative stress of bovine mammary epithelial cells in response to exogenous free fatty acids (FFA).ResultsExogenous FFA induced linear and quadratic decreases in activities of glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD), and increases in the contents of reactive oxygen species (ROS) and malondialdehyde (MDA). Protein abundance of LC3-phosphatidylethanolamine conjugate (LC3-II) and the number of autophagosomes and autolysosomes decreased in a dose-dependent manner, while protein abundance of p62 increased in cells challenged with FFA. Activation of autophagy via pre-treatment with Rap attenuated the FFA-induced ROS accumulation. Importantly, FFA inhibited protein abundance of NFE2L2 and the translocation of NFE2L2 into the nucleus. Knockdown of NFE2L2 by siRNA decreased protein abundance of LC3-II, while it increased protein abundance of p62. Furthermore, sulforaphane (SFN) pre-treatment attenuated the FFA-induced oxidative stress by activating NFE2L2-mediated autophagy.ConclusionsThe data suggested that NFE2L2-mediated autophagy is an important antioxidant mechanism in bovine mammary epithelial cells experiencing increased FFA loads.

Omega-3 polyunsaturated fatty acids ameliorated inflammatory response of mammary epithelial cells and mammary gland induced by lipopolysaccharide.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), essential fatty acids for humans and animals, have been reported to play a beneficial role in a variety of inflammatory diseases. In this study, we investigated the inhibitory effects and potential molecular mechanisms of n-3 PUFAs on the inflammatory response in lipopolysaccharide (LPS)-stimulated mammary alveolar cell line (MAC-T). Results showed that n-3 PUFAs could abate LPS-induced secretions of tumor necrosis factor-α, interleukin (IL)-6 and IL-1β in MAC-T cells through the nuclear transcription factor kappa B (NF-κB) signal pathway. Meanwhile, n-3 PUFA intervention attenuated histopathologic changes of mammary glands, the white blood cell number decrease, and the alkaline phosphatase level decrease in the serum of mice challenged by LPS. Furthermore, n-3 PUFA intervention improved the ecological structure of the flora in terms of the structural disorder of the non-significant dominant flora induced by LPS in mice. Collectively, both in vitro and in vivo experiments revealed that n-3 PUFAs have a positive effect on LPS-induced inflammatory response, which was possibly mediated by the NF-κB signaling pathway and the intestinal microbiota.

Substratum stiffness tunes membrane voltage in mammary epithelial cells.

Membrane voltage (Vm) plays a critical role in the regulation of several cellular behaviors, including proliferation, apoptosis and phenotypic plasticity. Many of these behaviors are affected by the stiffness of the underlying extracellular matrix, but the connections between Vm and the mechanical properties of the microenvironment are unclear. Here, we investigated the relationship between matrix stiffness and Vm by culturing mammary epithelial cells on synthetic substrata, the stiffnesses of which mimicked those of the normal mammary gland and breast tumors. Although proliferation is associated with depolarization, we surprisingly observed that cells are hyperpolarized when cultured on stiff substrata, a microenvironmental condition that enhances proliferation. Accordingly, we found that Vm becomes depolarized as stiffness decreases, in a manner dependent on intracellular Ca2+. Furthermore, inhibiting Ca2+-gated Cl- currents attenuates the effects of substratum stiffness on Vm. Specifically, we uncovered a role for cystic fibrosis transmembrane conductance regulator (CFTR) in the regulation of Vm by substratum stiffness. Taken together, these results suggest a novel role for CFTR and membrane voltage in the response of mammary epithelial cells to their mechanical microenvironment.

Latent TGF-β Activation Is a Hallmark of the Tenascin Family.

Transforming growth factor-β (TGF-β) isoforms are secreted as inactive complexes formed through non-covalent interactions between bioactive TGF-β entities and their N-terminal pro-domains called latency-associated peptides (LAP). Extracellular activation of latent TGF-β within this complex is a crucial step in the regulation of TGF-β activity for tissue homeostasis and immune cell function. We previously showed that the matrix glycoprotein Tenascin-X (TN-X) interacted with the small latent TGF-β complex and triggered the activation of the latent cytokine into a bioactive TGF-β. This activation most likely occurs through a conformational change within the latent TGF-β complex and requires the C-terminal fibrinogen-like (FBG) domain of the glycoprotein. As the FBG-like domain is highly conserved among the Tenascin family members, we hypothesized that Tenascin-C (TN-C), Tenascin-R (TN-R) and Tenascin-W (TN-W) might share with TN-X the ability to regulate TGF-β bioavailability through their C-terminal domain. Here, we demonstrate that purified recombinant full-length Tenascins associate with the small latent TGF-β complex through their FBG-like domains. This association promotes activation of the latent cytokine and subsequent TGF-β cell responses in mammary epithelial cells, such as cytostasis and epithelial-to-mesenchymal transition (EMT). Considering the pleiotropic role of TGF-β in numerous physiological and pathological contexts, our data indicate a novel common function for the Tenascin family in the regulation of tissue homeostasis under healthy and pathological conditions.

Role of long polar fimbriae type 1 and 2 in pathogenesis of mammary pathogenic Escherichia coli

Escherichia coli is a leading cause of bovine mastitis worldwide. The bacteria can rapidly grow in milk and elicit a strong lipopolysaccharide (LPS)/toll-like receptor-4 (TLR4)-dependent inflammatory response. Recently, the long polar fimbriae (LPF) were identified as a promising virulence factor candidate widely distributed in mammary pathogenic E. coli (MPEC) strains. Mammary pathogenic E. coli possess 2 lpf loci encoding LPF1 and LPF2, respectively. By deleting the major fimbrial subunit gene, lpfA, we found that both LPF1 and LPF2 contribute to MPEC adhesion, invasion, and biofilm formation in vitro. The lpf1A and lpf2A mutants showed reduced cytotoxicity in our in vitro cell infection model. Furthermore, we observed that LPF2 induced a mild TLR4-independent proinflammatory response. The median lethal dose (LD50) of both ∆lpf2A and ∆lpf1A∆lpf2A mutants to BALB/c mice increased by 0.38 and 0.15 logs, respectively, whereas that of wild-type strain MPJS13 was 8.69 logs. In contrast, LPF1 deficiency significantly enhanced the LPS/TLR4-mediated inflammatory response in mammary epithelial cells, and the LD50 of the mutant decreased to 8.18 logs. In conclusion, our data suggested that LPF are important in MPEC colonization of mammary cells and may provide a benefit to bacterial intracellular survival that induces persistent bovine mastitis.

Palmatine attenuates LPS-induced inflammatory response in mouse mammary epithelial cells through inhibiting ERK1/2, P38 and Akt/NF-кB signalling pathways.

Palmatine has a wide range of pharmacological effects and anti-inflammatory function. However, the effect of palmatine on LPS-induced inflammatory response of mammary epithelial cells has not been reported. In this research, we studied the anti-inflammatory mechanism of palmatine in EpH4-Ev (mouse mammary epithelial cells). EpH4-Ev cells were pre-treated with palmatine and then incubated with LPS. Cells were collected for examining production of pro-inflammatory mediators by qRT-PCR, and the related inflammatory signalling pathway was detected through immunofluorescence and Western blot. The results found that palmatine could significantly reduce the expression of IL-6, TNF-α, IL-1β and COX-2 in EpH4-Ev cells. Research on mechanisms found that palmatine could significantly inhibit the protein levels of p-Akt, p-P65, p-ERK1/2 and p-P38 in EpH4-Ev cells. In conclusion, these data suggested that palmatine inhibits inflammatory response in LPS-induced EpH4-Ev cells via down-regulating Akt/ NF-кB, ERK1/2 and P38 signalling pathways.

Acute phase protein expressions in secretory and cistern lining epithelium tissues of the dairy cattle mammary gland during chronic mastitis caused by staphylococci

BackgroundMastitis is the most common disease in dairy cattle and the costliest for the dairy farming industry, as it lowers milk yield and quality. Mastitis occurs as a result of interactions between microorganisms and the individual genetic predispositions of each animal. Thus, it is important to fully understand the mechanisms underlying these interactions. Elucidating the immune response mechanisms can determine which genetic background makes an animal highly resistant to mastitis. We analyzed the innate immune responses of dairy cows naturally infected with coagulase-positive staphylococci (CoPS; N = 8) or coagulase-negative staphylococci (CoNS; N = 7), causing persistent mastitis (after several failed treatments) vs. infection-free (i.e., healthy [H]; N = 8) dairy cows. The expressions of the acute phase protein genes serum amyloid A3 (SAA3), haptoglobin (HP), ceruloplasmin (CP) genes in the tissues most exposed to pathogens— mammary gland cistern lining epithelial cells (CLECs) and mammary epithelial cells (MECs)—were analyzed.ResultsWe found constitutive and extrahepatic expressions of the studied genes in both tissue types. HP expression in the MECs of the CoPS-infected group was higher than in the H group (p ≤ 0.05). Moreover, higher SAA3 expression in the CoPS and CoNS groups than in the H group (p = 0.06 and 0.08, respectively) was found. No differences between SAA3 and HP in CLECs were revealed, regardless of the pathogen type. However, higher expression of CP (p ≤ 0.05) in the CoPS group than in the H group was noted.ConclusionsThe expressions of selected acute phase proteins were similar between CLECs and MECs, which means that CLECs are not only a mechanical barrier but are also responsible for the biological immune response. Our findings agree with the results of other authors describing the immunological response of MECs during chronic mastitis, but the results for CLECs are novel.

Investigation of potential early key events and mode of action for 1,2-dichloroethane-induced mammary tumors in female rats.

1,2-dichloroethane (DCE or EDC) is a chlorinated hydrocarbon used as a chemical intermediate, including in the synthesis of polyvinyl chloride. Although DCE has induced tumors in both rats and mice, the overall weight-of-evidence suggests a lack of in vivo mutagenicity. The present study was conducted to explore a potential mode of action further for tumor formation in rat mammary tissue. Fischer 344 rats were exposed to target concentrations of 0 or 200 ppm of DCE vapors (6 hours/day, 7 days/week) for at least 28 days; 200 ppm represents a concentration of ~20% higher than that reported to induce mammary tumors. Endpoints examined included DNA damage (via Comet assay), glutathione (reduced, oxidized and conjugated), tissue DNA adducts, cell proliferation and serum prolactin levels. Exposure to DCE did not alter serum prolactin levels with consistent estrous stage, did not cause cell proliferation in mammary epithelial cells, nor result in histopathological alterations in the mammary gland. DNA adducts were identified, including the N7 -guanylethyl glutathione adduct, with higher adduct levels measured in liver (nontumorigenic target) compared with mammary tissue isolated from the same rats; no known mutagenic adducts were identified. DCE did not increase the Comet assay response in mammary epithelial cells, whereas DNA damage in the positive control (N-nitroso-N-methylurea) was significantly increased. Although the result of this study did not identify a specific mode of action for DCE-induced mammary tumors in rats, the lack of any exposure-related genotoxic responses further contributes to the weight-of-evidence suggesting that DCE is a nongenotoxic carcinogen.